The invention is in the field of Brassica juncea breeding (i.e., Brassica), specifically relating to the development of stable herbicide tolerant Brassica juncea lines, plants and plant parts. A method of producing stable herbicide tolerant Brassica juncea lines, plants and plant parts is also provided.
Several Brassica species are recognized as an increasingly important oilseed crop and a source of high quality protein meal in many parts of the world. The oil extracted from the seeds commonly contains a lesser concentration of endogenously formed saturated fatty acids than other vegetable oils and is well suited for use in the production of salad oil or other food products or in cooking or frying applications. The oil also finds utility in industrial applications. Additionally, the meal component of the seeds can be used as a nutritious protein concentrate for livestock.
The three primary Brassica species currently utilized for Brassica production and development are Brassica napus, Brassica rapa and Brassica juncea, each of which belong to the family Brassicaceae. Brassica juncea is currently grown as an oilseed in India and China. As Brassica juncea tolerates heat and drought conditions to a greater extent than Brassica napus and Brassica rapa, there is potential for Brassica juncea production in certain areas of the United States, Canada and Australia. Table 1 contains a comparative description of the general characteristics of Brassica napus, Brassica rapa and Brassica juncea compiling information from the Canola Council of Canada worldwide web site and the USDA circular number C857 by Albina Musil USDA1950C857 (1951).
Brassica juncea is commonly grown as a condiment mustard species in several countries including Canada, Hungary, Poland, Ukraine, China, Nepal and India. Mustard quality Brassica juncea is typically high in glucosinolate and high in erucic acid content, but is relatively low in oil content. Mustard seed can be used in whole seed or crushed form. Seed may be milled into flour or the oil may be extracted for use in cooking. High glucosinolate and high erucic acid types are quality variants within the same species, differing only in quality parameters. As a result, cross breeding between low and high glucosinolate or erucic acid genotypes are easily made.
Certain genotypes of Brassica juncea generally possess relatively low erucic acid levels in the oil and low glucosinolate levels in the meal. Therefore, certain commercial varieties of Brassica juncea may be developed that can be termed xe2x80x9cCANOLA(copyright)xe2x80x9d in accordance with the trademark of the Canola Council of Canada, which refers to forms of oilseed Brassica with erucic acid of  less than 2% in the oil and total glucosinolates of  less than 30 micromoles/gram of defatted meal
The genomic composition of canola species are as follows (FIG. 1). Brassica rapa, a diploid species, contains only the A (rapa) genome and has a genomic constitution of AA. Brassica napus is an amphidiploid with the rapa (A) and oleraceae (C) genomes and is listed as AACC. Brassica juncea is also an amphidiploid with the rapa (A) genome and the nigra (B) genome. Genetically, Brassica juncea is listed as AABB.
During pollen and ovule formation, the chromosomes within each genome will pair with their homologues (i.e., xe2x80x98Axe2x80x99 chromosomes will pair with xe2x80x98Axe2x80x99, xe2x80x98Bxe2x80x99 will pair with xe2x80x98Bxe2x80x99), and it is extremely rare to have pairing of A and B or A and C. This pairing may be forced by repeated crossing and careful selection of plant phenotype during breeding, although there is no expectation that a trait from one genome may be combined with a trait from the other genome.
Brassica sp. cultivars are developed through breeding programs that utilize techniques such as mass and recurrent selection, backcrossing, pedigree breeding and haploidy. Recurrent selection is used to improve populations of either self- or cross-pollinating Brassica. Through recurrent selection, a genetically variable population of heterozygous individuals is created by intercrossing several different parents. The best plants are selected based on individual superiority, outstanding progeny, or excellent combining ability. The selected plants are intercrossed to produce a new population in which further cycles of selection are continued. Various recurrent selection techniques are used to improve quantitatively inherited traits controlled by numerous genes.
Breeding programs use backcross breeding to transfer genes for a simply inherited, highly heritable trait into another line that serves as the recurrent parent. The source of the trait to be transferred is called the donor parent. After the initial cross, individual plants possessing the desired trait of the donor parent are selected and are crossed (backcrossed) to the recurrent parent for several generations. The resulting plant is expected to have the attributes of the recurrent parent and the desirable trait transferred from the donor parent. This approach has been used for breeding disease resistant phenotypes of many plant species. However, certain traits are difficult to transfer by backcross breeding because other attributes of the recurrent parent are linked to the desirable trait, and therefore it is difficult to develop a resulting plant with all of the attributes of the recurrent parent and the desirable trait transferred from the donor parent. Backcrossing has been used to transfer low erucic acid and low glucosinolate content into lines and breeding populations of Brassica.
Pedigree breeding and recurrent selection breeding methods are used to develop lines from breeding populations. Pedigree breeding starts with the crossing of two genotypes, each of which may have one or more desirable characteristics that is lacking in the other or which complements the other. If the two original parents do not provide all of the desired characteristics, other sources can be included in the breeding population. In the pedigree method, superior plants are selfed and selected in successive generations. In the succeeding generations the heterozygous condition gives way to homogeneous lines as a result of self-pollination and selection. Typically in the pedigree method of breeding five or more generations of selfing and selection is practiced: F1 to F2; F2 to F3; F3 to F4; F4 to F5, etc. For example, two parents that are believed to possess favorable complementary traits are crossed to produce an F1. An F2 population is produced by selfing one or several F1""s or by intercrossing two F1""s (i.e., sib mating). Selection of the best individuals may begin in the F2 population, and beginning in the F3 the best individuals in the best families are selected. Replicated testing of families can begin in the F4 generation to improve the effectiveness of selection for traits with low heritability. At an advanced stage of inbreeding (i.e., F6 and F7), the best lines or mixtures of phenotypically similar lines commonly are tested for potential release as new cultivars. Backcrossing may be used in conjunction with pedigree breeding; for example, a combination of backcrossing and pedigree breeding with recurrent selection has been used to incorporate blackleg resistance into certain cultivars of Brassica napus. 
Plants that have been self-pollinated and selected for type for many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny. If desired, the haploidy method can also be used to extract homogeneous lines. A cross between two different homozygous lines produces a uniform population of hybrid plants that may be heterozygous for many gene loci. A cross of two plants each heterozygous at a number of gene loci will produce a population of hybrid plants that differ genetically and will not be uniform.
The choice of breeding or selection methods depends on the mode of plant reproduction, the heritability of the trait(s) being improved, and the type of cultivar used commercially (e.g., F1 hybrid cultivar, pureline cultivar, etc.).
The invention is in the field of Brassica juncea (i.e. Brassica) breeding, specifically relating to the development of stable herbicide tolerant Brassica juncea lines, plants and plant parts. A method of producing stable herbicide tolerant Brassica juncea lines, plants and plant parts is also provided.
In the description and tables which follow a number of terms are used. In order to aid in a clear and consistent understanding of the specification the following definitions and evaluation criteria are provided.
Cotyledon. A cotyledon is a type of seed leaf that is contained on a plant embryo. A cotyledon contains the food storage tissues of the seed. The embryo is a small plant contained within a mature seed.
Cotyledon Length. The distance between the indentation at the top of the cotyledon and the point where the width of the petiole is approximately 4 mm.
Cotyledon Width. The width at the widest point of the cotyledon when the plant is at the two to three-leaf stage of development (mean of 50).
Fatty Acid Content: The typical percentages by weight of fatty acids present in the endogenously formed oil of the mature whole dried seeds are determined. During such determination, the seeds are crushed and are extracted as fatty acid methyl esters following reaction with methanol and sodium methoxide. Next the resulting ester is analyzed for fatty acid content by gas liquid chromatography using a capillary column which allows separation on the basis of the degree of unsaturation and fatty acid chain length. This procedure is described in the work of J. K. Daun et al. J. Amer. Oil Chem. Soc., 60: 1751 to 1754 (1983) which is herein incorporated by reference.
Flower Bud Location. A determination is made whether typical buds are disposed above or below the most recently opened flowers.
Glucosinolate Content. The total aliphatic glucosinolate content of the meal of the seeds is determined on the moisture free air-dried-oil-free solid meal as measured by the gas liquid chromatography method of the Canadian Grain Commission as is expressed micromoles per gram. Capillary gas chromatography of the trimethylsityl derivatives of extracted and purified desulfoglucosinolates with optimization to obtain optimum indole glucosinolate detection as described in xe2x80x9cProcedures of the Western Canada Canola/Rapeseed Recommending Committee Incorporated for the Evaluation and Recommendation for Registration of Canola/Rapeseed Candidate Cultivars in Western Canadaxe2x80x9d. 
Growth Habit. This refers to whether the Brassica is primarily a spring annual or winter annual type.
Herbicide Tolerance. Tolerance to various herbicides when applied at standard recommended application rates is expressed on a scale of 1 (highly tolerant), 2 (tolerant), or 3 (susceptible).
Leaf Morphology. Includes characteristics such as leaf attachment to stem, leaf color, leaf dentation, leaf margin hairiness. Often observed on first leaves and again when at least 6 leaves of the plant are completely developed.
Mutagenesis. Any one of many techniques known in the art to create or induce genetic mutations, including, without limitation, microspore mutagenesis as described in Swanson et al., Plant Cell Reports 7:83-87 (1989).
Oil Content. The typical percentage by weight oil present in the mature whole dried seeds is determined by ISO 10565:1993 Oilseeds Simultaneous determination of oil and waterxe2x80x94Pulsed NMR method.
Plant Height. The overall plant height at the end of flowering is observed (mean of 50).
Ploidy. This refers to whether the number of xe2x80x9cbasic setsxe2x80x9d of chromosomes (individual replicates of the same genome) exhibited by the cultivar is diploid (two sets) or amphidiploid (two sets each of two different genomes).
Resistance to Shattering. Resistance to silique shattering is observed at seed maturity and is expressed on a scale of 1 (poor) to 5 (excellent).
Seed Coat Color. The seed coat color of typical mature seeds is observed.